Novel Mutations in a Tissue Culture-Adapted Hepatitis C Virus Strain Improve Infectious-Virus Stability and Markedly Enhance Infection Kinetics

172

H epatitis C virus (HCV) infects an estimated 180 million people worldwide, and about 75% of newly infected patients progress toward a chronic infection, which constitutes a risk for severe liver diseases such as cirrhosis and hepatocarcinoma (1-4). HCV is a hepacivirus in the Flaviviridae family characterized by the error-prone replication and quasispecies dynamics typical of RNA viruses (2,(5)(6)(7). No vaccine is available to prevent HCV infections or disease, and the current standard of care treatment consists of the combination of pegylated alpha interferon (IFN-␣) and the purine nucleoside analogue ribavirin (1-␤-D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) (8-10). Type I interferons (IFNs) such as IFN-␣ are members of a family of cytokines that constitute key components of the innate immune response to viruses, and their induction results in an antiviral state of the cell and suppression of viral replication (11)(12)(13)(14). Ribavirin is currently used to treat a number of human viral infections, and it can display multiple mechanisms of action, including enhancement of Th1 antiviral immune responses, upregulation of IFN-stimulated genes (ISGs), depletion of GTP pools, or viral RNA mutagenesis (see reviews in references 15, 16 and 17). However, on average only about 60% of the chronically infected patients show a sustained virological response to treatment with IFN-␣ plus ribavirin that results in virus clearance (18-21). The molecular and physiological bases of the therapeutic activity of the current combination treatment with IFN-␣ plus ribavirin are poorly understood. It might be possible to establish new treatment protocols based on recently developed directly acting antiviral agents (DAAs), with or without .Highly variable viruses exploit a number of strategies to counteract selective constraints intended to prevent their replication (reviewed in reference 7). Mutations that confer resistance to DAAs generally map in the target protein or in a protein that interacts with the target (28-30). However, the complexity of the cellular IFN response pathway is expected to require greater diversity of viral resistance mutations. Current evidence suggests that the response of HCV to IFN-␣-based therapy is influenced by several host (i.e., interleukin-28B [IL-28B] gene polymorphisms) and viral genetic (i.e., viral genotype and population complexity) factors (31-37).The advent of cell culture systems for sustained replication of HCV offers new prospects to approach the evolutionary dynamics of HCV and the host cell-virus relationship, including the anti-HCV activity of IFN-␣. Using these systems, Garaigorta and Chisari documented that HCV-induced protein kinase R (PKR) phosphorylation inhibited translation of IFN-stimulated genes (ISGs) in infected hepatocytes (38). Here we describe the adaptation of the HCV replication system of genotype 2a (39-41) to perform serial passages of HCV in the Huh-7.5 hepatocyte cell line with sustained, efficient viral replication for at least 100 serial passages. This cell ...

Section: Discussionmentioning

confidence: 99%

Response of Hepatitis C Virus to Long-Term Passage in the Presence of Alpha Interferon: Multiple Mutations and a Common Phenotype

Perales

Beach

Gallego

et al. 2013

172

“…In terms of replication, the NS3-5B region is sufficient to sustain viral RNA synthesis (12), and each of the proteins derived from this polyprotein is also known to contribute to virus production (30,(35)(36)(37)(38)(39)(40)(41)(42)(44)(45)(46)(47)(48). Our previous studies have shown that the kinetics of cleavage at boundaries between the NS proteins plays a critical role in reg- , and G1911A refer to the NS3-5B polyprotein expressed in the second cistron for each construct; wt denotes wt versions of NS5A and NS4B, S2208I indicates the mutation in NS5A, and G1911A denotes the mutation in NS4B.…”

Section: Discussionmentioning

confidence: 99%

“…Cell imaging shows that the RC lies proximal to sites where virus assembly probably occurs, since double-stranded RNA and MAF colocalize with envelope proteins and core-coated lipid droplets (30)(31)(32); the latter structures are thought to represent both a repository and a transitionary step in the maturation of core from a monomeric protein to a capsid shell (33,34). In addition, it has been conclusively demonstrated that mutations in the NS proteins can affect the efficiency of virus particle production independently of any effect that these changes may have on RNA replication (30,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). Such data illustrate the tight link between viral RNA replication and virion assembly.…”

mentioning

confidence: 99%

Genetic Complementation of Hepatitis C Virus Nonstructural Protein Functions Associated with Replication Exhibits Requirements That Differ from Those for Virion Assembly

Herod

Schregel

Hinds

et al. 2014

Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components required for viral RNA replication lie in the NS3-5B region, while virion assembly requires expression of all virus components. Here, we have employed complementation systems to examine the role that HCV polyprotein precursors play in RNA replication and virion assembly. In a trans-complementation assay, an HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replicationdefective mutation in NS5A. However, this requirement for precursor expression was partially alleviated when a second functional copy of NS5A was expressed from an additional upstream cistron within the RNA to be rescued. In contrast, rescue of a virion assembly mutation in NS5A was more limited but exhibited little or no requirement for expression of functional NS5A as a precursor, even when produced in the context of a second replicating helper RNA. Furthermore, expression of NS5A alone from an additional cistron within a replicon construct gave greater rescue of virion assembly in cis than in trans. Combined with the findings of confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems colocalize, the results point to NS3-5A playing a role in facilitating the integration of nonstructural (NS) proteins into viral membrane-associated foci, with this representing an early stage in the steps leading to replication complex formation. The data further imply that HCV employs a minor virion assembly pathway that is independent of replication. IMPORTANCEIn hepatitis C virus-infected cells, replication is generally considered an absolute prerequisite for virus particle formation. Here we investigated the role that the viral protein NS5A has in both replication and particle assembly using complementation assays and microscopy. We found that efficient rescue of replication required NS5A to be expressed as part of a larger polyprotein, and this correlated with detection of NS5A at sites where replication occurred. In contrast, rescue of particle assembly did not require expression of NS5A within the context of a polyprotein. Interestingly, although only partial restoration of particle assembly was possible by complementation, that proportion that could be rescued benefitted from expressing NS5A from the same RNA being packaged. Collectively, these findings provide new insight into aspects of polyprotein function. They also support the existence of a minor virion assembly pathway that bypasses replication.

“…NS3-NS5B are essential for HCV RNA synthesis (25) and localize to replication complexes (RCs) embedded within cellular endoplasmic reticulum (ER) membranes (8,13,41,48). However, many of the nonstructural proteins are also engaged in the generation of infectious virions, with reports of NS3 (14,26,53), NS4A (38), NS4B (17,40), and NS5A (3,27,31,50,51) being essential for, or at least contributing to, this process. It is therefore becoming apparent that the assembly of infectious virus particles is a complex, multistep mechanism involving the majority of the HCV-encoded proteins.…”

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confidence: 99%

A Genetic Interaction between the Core and NS3 Proteins of Hepatitis C Virus Is Essential for Production of Infectious Virus

Jones

Atoom

Zhang

et al. 2011

By analogy to other members of the Flaviviridae family, the hepatitis C virus (HCV) core protein is presumed to oligomerize to form the viral nucleocapsid, which encloses the single-stranded RNA genome. Core protein is directed to lipid droplets (LDs) by domain 2 (D2) of the protein, and this process is critical for virus production. Domain 1 (D1) of core is also important for infectious particle morphogenesis, although its precise contribution to this process is poorly understood. In this study, we mutated amino acids 64 to 75 within D1 of core and examined the ability of these mutants to produce infectious virus. We found that residues 64 to 66 are critical for generation of infectious progeny, whereas 67 to 75 were dispensable for this process. Further investigation of the defective 64 to 66 mutant (termed JFH1 T -64-66) revealed it to be incapable of producing infectious intracellular virions, suggesting a fault during HCV assembly. Furthermore, isopycnic gradient analyses revealed that JFH1 T -64-66 assembled dense intracellular species of core, presumably representing nucleocapsids. Thus, amino acids 64 to 66 are seemingly not involved in core oligomerization/nucleocapsid assembly. Passaging of JFH1 T -64-66 led to the emergence of a single compensatory mutation (K1302R) within the helicase domain of NS3 that completely rescued its ability to produce infectious virus. Importantly, the same NS3 mutation abrogated virus production in the context of wild-type core protein. Together, our results suggest that residues 64 to 66 of core D1 form a highly specific interaction with the NS3 helicase that is essential for the generation of infectious HCV particles at a stage downstream of nucleocapsid assembly.Hepatitis C virus (HCV) typically establishes chronic infections of the liver that frequently lead to severe pathologies, including cirrhosis and hepatocellular carcinoma. The current combination therapy of pegylated alpha interferon (IFN-␣) and ribavirin is only partially effective and is associated with numerous side effects. Treatment for HCV is currently transitioning to the use of direct-acting antiviral (DAA) therapy, which has been specifically designed to target viral proteins essential for HCV replication (30). While early results indicate that DAA compounds show promise, a wider range of treatments targeting multiple aspects of the viral life cycle would offer improved therapeutic options to infected individuals. In this regard, a better understanding of HCV assembly could provide an alternate exploitable target.